Inflammation is the body's defense reaction to injuries such as those caused by mechanical damage, infection or antigenic stimulation. An inflammatory reaction may be expressed pathologically when inflammation is induced by an inappropriate stimulus such as an autoantigen, is expressed in an exaggerated manner or persists well after the removal of the injurious agents. Such inflammatory reaction may include the production of certain cytokines.
While the etiology of inflammation is poorly understood, considerable information has recently been gained regarding the molecular aspects of inflammation. This research has led to identification of certain cytokines which are believed to figure prominently in the mediation of inflammation. Cytokines are extracellular proteins that modify the behavior of cells, particularly those cells that are in the immediate area of cytokine synthesis and release. Tumor necrosis factors (TNFs) are a class of cytokines produced by numerous cell types, including monocytes and macrophages.
At least two TNFs have been previously described, specifically TNF alpha (TNF-α) and TNF beta (TNF-β or lymphotoxin), and each is active as a trimeric molecule and is believed to initiate cellular signaling by crosslinking receptors (Engelmann et al. (1990), J. Biol. Chem., 265:14497-14504).
Several lines of evidence implicate TNF-α and TNF-β as major inflammatory cytokines. These known TNFs have important physiological effects on a number of different target cells which are involved in inflammatory responses to a variety of stimuli such as infection and injury. The proteins cause both fibroblasts and synovial cells to secrete latent collagenase and prostaglandin E2 and cause osteocyte cells to stimulate bone resorption. These proteins increase the surface adhesive properties of endothelial cells for neutrophils. They also cause endothelial cells to secrete coagulant activity and reduce their ability to lyse clots. In addition they redirect the activity of adipocytes away from the storage of lipids by inhibiting expression of the enzyme lipoprotein lipase. TNFs also cause hepatocytes to synthesize a class of proteins known as “acute phase reactants,” which act on the hypothalamus as pyrogens (Selby et al. (1988), Lancet, 1(8583):483; Starnes, Jr. et al. (1988), J. Clin. Invest., 82:1321; Oliff et al. (1987), Cell, 50:555; and Waage et al. (1987), Lancet, 1(8529):355). Additionally, preclinical results with various predictive animal models of inflammation, including rheumatoid arthritis, have suggested that inhibition of TNF can have a major impact on disease progression and severity (Dayer et al. (1994), European Cytokine Network, 5(6):563-571 and Feldmann et al. (1995), Annals Of The New York Academy Of Sciences, 66:272-278). Moreover, recent preliminary human clinical trials in rheumatoid arthritis with inhibitors of TNF have shown promising results (Rankin et al. (1995), British Journal Of Rheumatology, 3(4):4334-4342; Elliott et al. (1995), Lancet, 344:1105-1110; Tak et al. (1996), Arthritis and Rheumatism, 39:1077-1081; and Paleolog et al. (1996), Arthritis and Rheumatism, 39:1082-1091).
Protein inhibitors of TNF are disclosed in the art. EP 308 378 reports that a protein derived from the urine of fever patients has a TNF inhibiting activity. The effect of this protein is presumably due to a competitive mechanism at the level of the receptors. EP 308 378 discloses a protein sufficiently pure to be characterized by its N-terminus. The reference, however, does not teach any DNA sequence or a recombinantly-produced TNF inhibitor.
Recombinantly-produced TNF inhibitors have also been taught in the art. For example, EP 393 438 and EP 422 339 teach the amino acid and nucleic acid sequences of a mature, recombinant human “30 kDa TNF inhibitor” (also known as a p55 receptor and as sTNFR-I) and a mature, recombinant human “40 kDa inhibitor” (also known as a p75 receptor and as sTNFR-II) as well as modified forms thereof, e.g., fragments, functional derivatives and variants. EP 393 438 and EP 422 339 also disclose methods for isolating the genes responsible for coding the inhibitors, cloning the gene in suitable vectors and cell types, and expressing the gene to produce the inhibitors. Mature recombinant human 30 kDa TNF inhibitor and mature recombinant human 40 kDa TNF inhibitor have been demonstrated to be capable of inhibiting TNF (EP 393 438, EP 422 339, PCT Publication No. WO 92/16221 and PCT Publication No. WO 95/34326).
sTNFR-I and sTNFR-II are members of the nerve growth factor/TNF receptor superfamily of receptors which includes the nerve growth factor receptor (NGF), the B cell antigen CD40, 4-1BB, the rat T-cell antigen MRC OX40, the Fas antigen, and the CD27 and CD30 antigens (Smith et al. (1990), Science, 248:1019-1023). The most conserved feature amongst this group of cell surface receptors is the cysteine-rich extracellular ligand binding domain, which can be divided into four repeating motifs of about forty amino acids and which contains 4-6 cysteine residues at positions which are well conserved (Smith et al. (1990), supra).
EP 393 438 further teaches a 40 kDa TNF inhibitor Δ51 and a 40 kDa TNF inhibitor Δ53, which are truncated versions of the full-length recombinant 40 kDa TNF inhibitor protein wherein 51 or 53 amino acid residues, respectively, at the carboxyl terminus of the mature protein are removed. Accordingly, a skilled artisan would appreciate that the fourth domain of each of the 30 kDa TNF inhibitor and the 40 kDa inhibitor is not necessary for TNF inhibition. In fact various groups have confirmed this understanding. Domain-deletion derivatives of the 30 kDa and 40 kDa TNF inhibitors have been generated, and those derivatives without the fourth domain retain full TNF binding activity while those derivatives without the first, second or third domain, respectively, do not retain TNF binding activity (Corcoran et al. (1994), Eur. J. Biochem., 223:831-840; Chih-Hsueh et al. (1995), The Journal of Biological Chemistry, 270(6):2874-2878; and Scallon et al. (1995), Cytokine, 7(8):759-770).
Due to the relatively low inhibition of cytotoxicity exhibited by the 30 kDa TNF inhibitor and 40 kDa TNF inhibitor (Butler et al. (1994), Cytokine, 6(6):616-623), various groups have generated dimers of TNF inhibitor proteins (Butler et al. (1994), supra; and Martin et al. (1995), Exp. Neurol., 131:221-228). However, the dimers may generate an antibody response (Martin et al. (1995), supra; and Fisher et al. (1996), The New England Journal of Medicine, 334(26):1697-1702).
It is an object of the present invention to provide functionally active truncated sTNFRs. This and other objects of the present invention will become apparent from the description hereinafter.